This invention relates to cardiovascular diseases.
The vascular system contains vessels that are morphologically and functionally distinct. For example, arteries carry oxygenated blood at high pressure from the heart, while veins serve as capacitance vessels for blood return. While some of the differences within the vascular system occur after the onset of function, a complete vascular loop, formed by the trunk artery and vein, is required to accommodate the output of the first heart beat. The major trunk vessels, the aorta and the axial vein, of vertebrate embryos are formed by the coalescence of scattered mesodermal angioblasts into simple endothelial tubes by a process termed xe2x80x9cvasculogenesis,xe2x80x9d which is distinguished from xe2x80x9cangiogenesis,xe2x80x9d the formation of vessels by sprouting and remodeling (Fishman, xe2x80x9cAssembly of blood vessels in the embryos,xe2x80x9d Lippincott-Raven Publishers, Philadelphia, 1996; Folkman et al., Cell 87:1153-1155, 1996; Risau, Nature 386:671-674, 1997; Yancopolos et al., Cell 93:661-664, 1998).
The aorta, the main trunk of the systemic arterial network, is subject to several congenital and acquired disorders, that may lead to severe complications in infancy and adulthood. Coarctation of the aorta is one of the most common human congenital cardiovascular diseases. In coarctation, a discrete, localized vascular malformation partially obstructs the descending aorta, the major artery to the body, and most frequently occurs distal to the origins of vessels supplying the head and arms. Its effects often become more physiologically severe at birth, when closure of the ductus can exacerbate the restriction to aortic blood flow. As a consequence of coarctation, affected individuals suffer from high blood pressure in the upper extremities and head, and from low pressure in the trunk and legs. Survival often depends on the development of collateral blood vessels, which facilitate blood circulation in a manner so as to bypass the lesion.
Another serious cardiovascular disease that affects large numbers of individuals is atherosclerosis. This condition is characterized by the deposition of lipids in the intima of large and medium-sized arteries. Such deposits are associated with fibrosis and calcification.
The gridlock mutation (grlm145) is a recessive mutation that was identified in the zebrafish system and that causes a focal vascular malformation resembling coarctation of the aorta in humans. In grlm145 mutant embryos, fluorescent dextran injected into the heart, to outline patent vessels, is blocked at the origin of the dorsal aorta at the region where the two anterior dorsal aortae merge to form the single aorta of the trunk. The fluorescent beads circulated normally in the head (Weinstein et al., Nature Medicine 1:1143-1147, 1995). Thus, the grl mutant embryos lack circulation to the trunk.
The invention provides a novel gene, gridlock, and its encoded protein. gridlock plays a role in vascular development and modeling, and a mutation in gridlock has been associated with an aortic arch disease, coarctation. Thus, gridlock nucleic acid molecules and polypeptides can be used in methods of diagnosing, treating, and preventing gridlock-related diseases and conditions, such as aortic arch diseases.
In one aspect, the invention provides a substantially pure gridlock polypeptide. In preferred embodiments of this aspect, the gridlock polypeptide includes the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:4, or includes an amino acid sequence that is substantially identical to the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:4. In other preferred embodiments of this aspect, the polypeptide is derived from a mammal, e.g., a human.
In another aspect, the invention provides a substantially pure nucleic acid molecule, e.g., DNA, including a sequence encoding a gridlock polypeptide. In a preferred embodiment of this aspect, the nucleic acid molecule encodes a human gridlock polypeptide. In other preferred embodiments of this aspect, the nucleic acid molecule encodes a polypeptide including the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:4, or encodes a polypeptide including an amino sequence that is substantially identical to the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:4. In other preferred embodiments of this aspect, the nucleic acid molecule includes a nucleotide sequence that is substantially identical to the nucleotide sequence of SEQ ID NO:1 or SEQ ID NO:3.
In a related aspect, the invention provides a nucleic acid molecule having at least 55% nucleic acid sequence identity to a sequence encoding a gridlock polypeptide or a fragment thereof, where the fragment includes at least six amino acids and the nucleic acid molecule hybridizes under high stringency conditions to at least a portion of a gridlock nucleic acid molecule, and does not hybridize under high stringency conditions to a nucleic acid molecule encoding a hairy-related protein family member. In a preferred embodiment of this aspect, the nucleic acid molecule has 100% complementarity to a nucleic acid molecule encoding a gridlock polypeptide or a fragment thereof including at least six amino acids, and the nucleic acid molecule hybridizes under high stringency conditions to at least a portion of a gridlock nucleic acid molecule, and does not hybridize under high stringency conditions to a nucleic acid molecule encoding a hairy-related protein family member.
In a related aspect, the invention provides a nucleic acid molecule including a sequence that is antisense to a gridlock coding strand or a fragment thereof. In a preferred embodiment of this aspect, the antisense sequence is specific for a mutated gridlock coding region.
In another related aspect, the invention provides a vector, e.g., a gene therapy vector including a gridlock nucleic acid molecule, and a cell including this vector.
In other aspects, the invention provides a non-human transgenic animal, e.g., a zebrafish, including a gridlock nucleic acid molecule, and a non-human animal having a knockout mutation in one or both alleles encoding a gridlock polypeptide. The invention also provides a cell from a non-human animal having a knockout mutation in one or both alleles encoding a gridlock polypeptide.
In other aspects, the invention provides a probe for analyzing the gridlock nucleic acid molecules of an animal, the probe having at least 55% nucleic acid sequence identity to a sequence encoding a gridlock polypeptide or a fragment thereof. The fragment includes at least six amino acids and the probe hybridizes under high stringency conditions to at least a portion of a gridlock nucleic acid molecule, and does not hybridize under high stringency conditions to a nucleic acid molecule encoding a hairy-related protein family member. In a preferred embodiment, the probe has 100% complementarity to a nucleic acid molecule encoding a gridlock polypeptide or a fragment thereof including at least six amino acids, and the probe hybridizes under high stringency conditions to at least a portion of a gridlock nucleic acid molecule, and does not hybridize under high stringency conditions to a nucleic acid molecule encoding a hairy-related protein family member.
In another aspect, the invention provides an antibody that specifically binds to a gridlock polypeptide, e.g., a polypeptide including the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:4.
In another aspect, the invention provides a method of detecting the presence of a gridlock polypeptide in a sample. The method involves contacting the sample with an antibody that specifically binds to a gridlock polypeptide and assaying for binding of the antibody to the polypeptide.
In another aspect, the invention provides a method of detecting the presence of a mutant gridlock polypeptide in a sample. The method involves contacting the sample with an antibody that specifically binds to a mutant gridlock polypeptide and assaying for binding of the antibody to the mutant polypeptide.
In another aspect, the invention provides a method of diagnosing an increased likelihood of developing a gridlock-related disease or condition in a test subject. The method involves analyzing nucleic acid molecules of the test subject to determine whether the test subject contains a mutation in a gridlock gene. The presence of the mutation is an indication that the test subject has an increased likelihood of developing a gridlock-related disease. In preferred embodiments of this aspect, the test subject is a mammal, e.g, a human. In other preferred embodiments of this aspect, the method includes the step of using nucleic acid molecule primers specific for the gridlock gene for nucleic acid molecule amplification by the polymerase chain reaction, or includes the step of sequencing gridlock nucleic acid molecules from the test subject. In another preferred embodiment of this aspect, the analyzing is carried out by restriction fragment length polymorphism (RFLP) analysis. In another preferred embodiment of this aspect, the disease or condition is coarctation of the aorta, interrupted aortic arch disease, or atherosclerosis.
In another aspect, the invention provides a kit for the analysis of a gridlock nucleic acid molecule. The kit includes a nucleic acid molecule probe for analyzing the gridlock nucleic acid molecules of a test subject.
In another aspect, the invention provides a kit for the analysis of a gridlock nucleic acid molecule. The kit includes antibodies for analyzing the gridlock protein of a test subject.
In another aspect, the invention provides a method for preventing or ameliorating the effect of a gridlock deficiency by administering to a subject having a gridlock deficiency an expression vector including a nucleic acid molecule encoding a functional gridlock polypeptide. The nucleic acid molecule is operably linked to a promoter and the gridlock polypeptide is sufficient to prevent or ameliorate the effect of the gridlock deficiency.
In a related aspect, the invention provides a method for preventing or ameliorating the effect of a gridlock deficiency by administering a functional gridlock polypeptide to a subject having a gridlock deficiency. The gridlock polypeptide is sufficient to prevent or ameliorate the effect of the gridlock deficiency.
In a further aspect, the invention provides a method for preventing or ameliorating the effect of a gridlock excess by administering an antisense gridlock molecule to a subject having a gridlock excess. The antisense gridlock molecule is sufficient to prevent or ameliorate the effect of the gridlock excess.
In a related aspect, the invention provides a method for preventing or ameliorating the effect of a gridlock excess by administering a gridlock antibody to a subject having a gridlock excess. The gridlock antibody is sufficient to prevent or ameliorate the effect of the gridlock excess.
In another aspect, the invention provides a method for identifying a compound that modulates the expression or activity of a gridlock nucleic acid molecule or polypeptide. The method involves contacting the gridlock nucleic acid molecule or polypeptide with the compound, and determining the effect of the compound on the gridlock expression or activity.
By xe2x80x9cpolypeptidexe2x80x9d or xe2x80x9cpolypeptide fragmentxe2x80x9d is meant a chain of two or more amino acids, regardless of any post-translational modification (e.g., glycosylation or phosphorylation), constituting all or part of a naturally or non-naturally occurring polypeptide. By xe2x80x9cpost-translational modificationxe2x80x9d is meant any change to a polypeptide or polypeptide fragment during or after synthesis. Post-translational modifications can be produced naturally (such as during synthesis within a cell) or generated artificially (such as by recombinant or chemical means). A xe2x80x9cproteinxe2x80x9d can be made up of one or more polypeptides.
By xe2x80x9cgridlock,xe2x80x9d xe2x80x9cgridlock protein,xe2x80x9d or xe2x80x9cgridlock polypeptidexe2x80x9d is meant a polypeptide that has at least 45%, preferably at least 60%, more preferably at least 75%, and most preferably at least 90% amino acid sequence identity to the sequence of the human or the zebrafish gridlock polypeptides shown in FIG. 2 (SEQ ID NO:2 and SEQ ID NO:4, respectively). A polypeptide having at least 85% amino acid identity to the bHLH or at least 55% amino acid identity to the Orange domains of the human or the zebrafish gridlock polypeptides shown in FIG. 2 (SEQ ID NO:2 and SEQ ID NO:4, respectively) can be considered a gridlock polypeptide. Most preferably, such a polypeptide has at least 95% amino acid identity in the bHLH domain or at least 90% amino acid identity in the Orange domain to the human or the zebrafish gridlock polypeptides shown in FIG. 2 (SEQ ID NO:2 and SEQ ID NO:4, respectively). Polypeptide products from splice variants of gridlock gene sequences and gridlock genes containing mutations (e.g., the grlm145 mutation) are also included in this definition. Preferably, a gridlock polypeptide contains a xe2x80x9cYRPWxe2x80x9d motif. A gridlock polypeptide as defined herein plays a role in vascular development and modeling. It can be used as an early marker of vasculogenesis or angioblast cell fate determination, as well as an artery-specific, for example, an aorta-specific, marker later in development or in adults.
By a xe2x80x9cgridlock nucleic acid moleculexe2x80x9d is meant a nucleic acid molecule, such as a genomic DNA, cDNA, or RNA (e.g., mRNA) molecule, that encodes gridlock, a gridlock protein, a gridlock polypeptide, or a portion thereof, as defined above.
The term xe2x80x9cidentityxe2x80x9d is used herein to describe the relationship of the sequence of a particular nucleic acid molecule or polypeptide to the sequence of a reference molecule of the same type. For example, if a polypeptide or nucleic acid molecule has the same amino acid or nucleotide residue at a given position, compared to a reference molecule to which it is aligned, there is said to be xe2x80x9cidentityxe2x80x9d at that position. The level of sequence identity of a nucleic acid molecule or a polypeptide to a reference molecule is typically measured using sequence analysis software with the default parameters specified therein, such as the introduction of gaps to achieve an optimal alignment (e.g., Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705, BLAST, or PILEUP/PRETTYBOX programs). These software programs match identical or similar sequences by assigning degrees of identity to various substitutions, deletions, or other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine, valine, isoleucine, and leucine; aspartic acid, glutamic acid, asparagine, and glutamine; serine and threonine; lysine and arginine; and phenylalanine and tyrosine.
A nucleic acid molecule or polypeptide is said to be xe2x80x9csubstantially identicalxe2x80x9d to a reference molecule if it exhibits, over its entire length, at least 51%, preferably at least 55%, 60%, or 65%, and most preferably 75%, 85%, 90%, or 95% identity to the sequence of the reference molecule. For polypeptides, the length of comparison sequences is at least 16 amino acids, preferably at least 20 amino acids, more preferably at least 25 amino acids, and most preferably at least 35 amino acids. For nucleic acid molecules, the length of comparison sequences is at least 50 nucleotides, preferably at least 60 nucleotides, more preferably at least 75 nucleotides, and most preferably at least 110 nucleotides.
A gridlock nucleic acid molecule or gridlock polypeptide is xe2x80x9canalyzedxe2x80x9d or subject to xe2x80x9canalysisxe2x80x9d if a test procedure is carried out on it that allows the determination of its biological activity or whether it is wild type or mutated. For example, one can analyze the gridlock genes of an animal (e.g., a human or a zebrafish) by amplifying genomic DNA of the animal using the polymerase chain reaction, and then determining whether the amplified DNA contains a mutation, e.g., by nucleotide sequence or restriction fragment analysis.
By xe2x80x9cprobexe2x80x9d or xe2x80x9cprimerxe2x80x9d is meant a single-stranded DNA or RNA molecule of defined sequence that can base pair to a second DNA or RNA molecule that contains a complementary sequence (xe2x80x9ctargetxe2x80x9d). The stability of the resulting hybrid depends upon the extent of the base pairing that occurs. This stability is affected by parameters such as the degree of complementarity between the probe and target molecule, and the degree of stringency of the hybridization conditions. The degree of hybridization stringency is affected by parameters such as the temperature, salt concentration, and concentration of organic molecules, such as formamide, and is determined by methods that are well known to those skilled in the art. Probes or primers specific for gridlock nucleic acid molecules, preferably, have greater than 45% sequence identity, more preferably at least 55-75% sequence identity, still more preferably at least 75-85% sequence identity, yet more preferably at least 85-99% sequence identity, and most preferably 100% sequence identity to the sequences designated as SEQ ID NO:1 or SEQ ID NO:3. Probes can be detectably-labeled, either radioactively or non-radioactively, by methods that are well-known to those skilled in the art. Probes can be used for methods involving nucleic acid hybridization, such as nucleic acid sequencing, nucleic acid amplification by the polymerase chain reaction, single stranded conformational polymorphism (SSCP) analysis, restriction fragment polymorphism (RFLP) analysis, Southern hybridization, northern hybridization, in situ hybridization, electrophoretic mobility shift assay (EMSA), and other methods that are well known to those skilled in the art.
A molecule, e.g., an oligonucleotide probe or primer, a gene or fragment thereof, a cDNA molecule, a polypeptide, or an antibody, can be said to be xe2x80x9cdetectably-labeledxe2x80x9d if it is marked in such a way that its presence can be directly identified in a sample. Methods for detectably-labeling molecules are well known in the art and include, without limitation, radioactive labeling (e.g., with an isotope, such as 32P or 35S) and nonradioactive labeling (e.g., with a fluorescent label, such as fluorescein).
By a xe2x80x9csubstantially pure polypeptidexe2x80x9d is meant a polypeptide (or a fragment thereof) that has been separated from proteins and organic molecules that naturally accompany it. Typically, a polypeptide is substantially pure when it is at least 60%, by weight, free from the proteins and naturally-occurring organic molecules with which it is naturally associated. Preferably, the polypeptide is a gridlock polypeptide that is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight, pure. A substantially pure gridlock polypeptide can be obtained, for example, by extraction from a natural source (e.g., isolated aorta or vascular tissue), by expression of a recombinant nucleic acid molecule encoding a gridlock polypeptide, or by chemical synthesis. Purity can be measured by any appropriate method, e.g., by column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.
A polypeptide is substantially free of naturally associated components when it is separated from those proteins and organic molecules that accompany it in its natural state. Thus, a protein that is chemically synthesized or produced in a cellular system different from the cell in which it is naturally produced is substantially free from its naturally associated components. Accordingly, substantially pure polypeptides not only include those derived from eukaryotic organisms, but also those synthesized in E. coli or other prokaryotes.
An antibody is said to xe2x80x9cspecifically bindxe2x80x9d to a polypeptide if it recognizes and binds to the polypeptide (e.g., a gridlock polypeptide), but does not substantially recognize and bind to other molecules (e.g., non-gridlock related polypeptides) in a sample, e.g., a biological sample, that naturally includes the polypeptide.
By xe2x80x9chigh stringency conditionsxe2x80x9d is meant conditions that allow hybridization comparable with the hybridization that occurs using a DNA probe of at least 500 nucleotides in length, in a buffer containing 0.5 M NaHPO4, pH 7.2, 7% SDS, 1 mM EDTA, and 1% BSA (fraction V), at a temperature of 65xc2x0 C., or a buffer containing 48% formamide, 4.8xc3x97 SSC, 0.2 M Tris-Cl, pH 7.6, 1xc3x97 Denhardt""s solution, 10% dextran sulfate, and 0.1% SDS, at a temperature of 42xc2x0 C. (These are typical conditions for high stringency northern or Southern hybridizations.) High stringency hybridization is also relied upon for the success of numerous techniques routinely performed by molecular biologists, such as high stringency PCR, DNA sequencing, single strand conformational polymorphism analysis, and in situ hybridization. In contrast to northern and Southern hybridizations, these techniques are usually performed with relatively short probes (e.g., usually 16 nucleotides or longer for PCR or sequencing and 40 nucleotides or longer for in situ hybridization). The high stringency conditions used in these techniques are well known to those skilled in the art of molecular biology, and examples of them can be found, for example, in Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, New York, N.Y., 1998, which is hereby incorporated by reference.
By a xe2x80x9ctransgenexe2x80x9d is meant a DNA molecule that is inserted by artifice into a cell (e.g., the nuclear genome of a cell), and is incorporated into the genome of an organism that develops from the cell. Such a transgene can be partly or entirely heterologous (i.e., foreign) to the transgenic organism, or can be a gene that is homologous to an endogenous gene of the organism. An organism or animal (e.g., a mammal, such as a mouse, rat, or goat) can be said to be xe2x80x9ctransgenicxe2x80x9d if it developed from a cell that had a transgene inserted into it by artifice.
By a xe2x80x9cknockout mutationxe2x80x9d is meant an artificially-induced alteration in a nucleic acid molecule (created by recombinant DNA technology or deliberate exposure to a mutagen) that reduces the biological activity of the polypeptide normally encoded therefrom by at least 80% relative to the unmutated gene. The mutation can be, without limitation, an insertion, deletion, frameshift mutation, or a missense mutation. A xe2x80x9cknockout animalxe2x80x9d is preferably a mammal, and more preferably a mouse, containing a knockout mutation, as defined above.
By xe2x80x9ctransformationxe2x80x9d is meant any method for introducing foreign molecules (e.g., nucleic acid molecules) into a cell. Lipofection, DEAE-dextran-mediated transfection, microinjection, protoplast fusion, calcium phosphate precipitation, retroviral delivery, electroporation, and biolistic transformation are just a few of the many transformation methods that are well known to those skilled in the art that can be used in the invention. For example, biolistic transformation is a method for introducing foreign molecules into a cell using velocity-driven microprojectiles such as tungsten or gold particles. Such methods can include helium-driven, air-driven, and gunpowder-driven techniques. Biolistic transformation can be applied to the transformation or transfection of a wide variety of cell types, intracellular organelles, and intact tissues including, without limitation, mitochondria, chloroplasts, bacteria, yeast, fungi, algae, animal tissue, and cultured cells.
By a xe2x80x9ctransformed cell,xe2x80x9d xe2x80x9ctransfected cell,xe2x80x9d or xe2x80x9ctransduced cell,xe2x80x9d is meant a cell (or a descendent of a cell) into which a DNA molecule encoding a polypeptide has been introduced, by means of recombinant DNA techniques.
By a xe2x80x9cpromoterxe2x80x9d is meant a minimal nucleic acid sequence element sufficient to direct transcription. If desired, constructs of the invention can include promoter elements that are sufficient to render promoter-dependent gene expression controllable in a cell type-specific, tissue-specific, or temporal-specific manner, or inducible by external signals or agents. Such elements can be located in the 5xe2x80x2, 3xe2x80x2, or intron regions of a gene.
By an xe2x80x9coperably linked sequencexe2x80x9d is meant that a gene and one or more regulatory sequences are connected in such a way as to permit gene expression when the appropriate molecules (e.g., transcriptional activator proteins) are bound to the regulatory sequences.
By an xe2x80x9cantisense molecule,xe2x80x9d as used herein in reference to nucleic acid molecules, is meant a nucleic acid molecule having a sequence that is complementary to at least 75 nucleotides, and preferably at least 100, 150, or 200 nucleotides, of the coding strand of a gene, such as a gridlock gene. An antisense nucleic acid molecule can be, for example, capable of preferentially lowering the production of a mutant gridlock polypeptide encoded by a mutant gridlock gene.
By xe2x80x9cmissense mutationxe2x80x9d is meant a substitution of one purine or pyrimidine base (i.e., A, T, G, or C) by another within a nucleic acid molecule, such that the resulting new codon encodes an amino acid that is distinct from the amino acid originally encoded by the wild type codon.
By xe2x80x9cframeshift mutationxe2x80x9d is meant an insertion or deletion of at least one nucleotide within a polynucleotide coding sequence. A frameshift mutation alters the codon reading frame at or downstream from the mutation site. Such a mutation results in either the substitution of an encoded wild type amino acid sequence by a novel amino acid sequence, or a premature termination of an encoded polypeptide, due to the creation of a stop codon, or both.
By xe2x80x9csamplexe2x80x9d is meant a tissue biopsy, amniotic fluid, cell, blood, serum, urine, stool, or other specimen obtained from a patient or test subject. The sample can be analyzed to detect a mutation in a gridlock gene, or expression levels of a gridlock gene, by methods that are known in the art. For example, methods such as sequencing, single-strand conformational polymorphism (SSCP) analysis, or restriction fragment length polymorphism (RFLP) analysis of PCR products derived from a patient sample can be used to detect a mutation in a gridlock gene; ELISA can be used to measure levels of gridlock polypeptide; and PCR can be used to measure the level of a gridlock nucleic acid molecule.
By xe2x80x9cgridlock-related diseasexe2x80x9d or xe2x80x9cgridlock-related conditionxe2x80x9d is meant a disease or condition that results from inappropriately high or low expression of a gridlock gene, or a mutation in a gridlock gene that alters the biological activity of a gridlock nucleic acid molecule or polypeptide. gridlock-related diseases and conditions can arise in any vascular tissue in which gridlock is expressed during prenatal or post-natal life. gridlock-related diseases and conditions can include congenital cardiovascular diseases (e.g., congenital dysmorphogenesis) and cardiovascular disease acquired in adulthood, such as atherosclerosis. By xe2x80x9ccongenital cardiovascular diseasexe2x80x9d is meant a disease associated with the abnormal formation of the heart or blood vessels. A patient suffering from a congenital cardiovascular disease, for example, an aortic arch disease, such as interrupted aortic arch disease or coarctation of the aorta, may display constriction of the aorta.
The invention provides several advantages. For example, it provides methods and reagents that can be used in the diagnosis and treatment of diseases caused by vascular malformation or dysfunction. These disorders include congenital cardiovascular diseases, such as interrupted arch diseases, aortic arch diseases, e.g., coarctation of the aorta, and cardiovascular diseases that occur in adulthood, such as atherosclerosis, hypertension, and coronary artery disease. These disorders can be treated, using the methods described herein, in a variety of ways including, for example, small molecule therapy, gene therapy, antisense oligonucleotide therapy, and protein replacement therapy. Additionally, the nucleic acid molecules and polypeptides of the invention have diagnostic/disease management applications, for example, use as prognostic markers or predisposition indicators of cardiovascular diseases.